S1P Receptor Targeted Drugs
S1P is an autocrine and paracrine signaling small molecule that impacts proliferation, survival and migration of endothelial cells, mural cells (i.e. vascular smooth muscle cells and pericytes), osteoblasts, and osteoblastic precursors through a family of high-affinity G protein-coupled receptors (S1P1-5). Selectively targeting a subset of S1P receptors with agonists and antagonist compounds (with longer bioactive half-lives than native S1P in vivo), one can control different biological responses. For example, recent reports suggest selective activation of S1P1 and S1P3 receptors via a synthetic analog of S1P, FTY720, promotes the recirculation of osteoclast precursor monocytes from the bone surface, an effect that ameliorates bone loss in models of postmenopausal osteoporosis. Furthermore, FTY720 treatment demonstrates enhanced CXCR4-mediated migration of endothelial progenitor cells and homing of bone marrow progenitors in hindlimb ischemia models. Recent discoveries of smooth muscle cell phenotype regulation in large arteries suggest possible synergies between S1P1 receptors and S1P3 receptors, both targets of FTY720. Specifically, daily injections of S1P1/S1P3 antagonist (VPC44116) significantly decreased smooth muscle proliferation and migration. Thus, FTY720 as a single bioactive factor has multiple cellular targets making it an attractive molecule for strategies to improve graft-host integration where multiple biological processes can be simultaneously augmented to address a central limitation, poor vascularization.
It has been shown that sustained release of FTY720 from two-dimensional biodegradable films (1:200 wt/wt) of 50:50 poly-lactic-co-glycolic acid (PLAGA) in the mouse dorsal skinfold window chamber promotes formation of new arterioles and structural enlargement of existing arterioles. This pattern of FTY720-induced microvascular remodeling increases the number and diameter of microvessels, a therapeutic response that is critical for successful integration of allograft implants in vivo. In addition, implantation of 3D PLAGA scaffolds delivering FTY720 to critical size calvarial bone defects significantly increases osseous tissue ingrowth and the proportion of mature smooth muscle cell-invested microvessels within the bony defect.
The G-protein coupled signaling pathway of S1P receptors has been shown to enhance cell motility, proliferation, and survival due to S1P stimulation. S1P is secreted by several types of cells including mast cells, macrophages, platelets, and endothelial cells into the blood flow in nanomolar plasma concentrations. In areas of endothelial injury, a higher concentration of S1P is released by activated platelets to aid in wound healing. Thus, S1P is thought to possess significant angiogenic and arteriogenic properties including mural cell recruitment to newly-formed vessels and stimulation of SMC differentiation, proliferation, and migration. S1P also reduces oxygen and nutrient-deprived cell death.
Fingolimod (FTY720) is a synthetic compound that acts as an agonist of the S1P1, S1P3, S1P4, and S1P5 receptors when phosphorylated into FTY720P. Due to its structural similarity with S1P, FTY720 shares many of the effects of natural S1P and thus acts as S1P analog. FTY720 was shown to stimulate the angiogenic activity and neovascularization of cultured cells. Other studies have shown that FTY720 prolongs allograft survival by preventing perivascular inflammation associated with chronic transplant rejection. Additionally, due to FTY720's rapid initial adsorption and exceptionally long half-life of approximately 7 days, the blood concentration of FTY720 remains relatively stable after administration. Native S1P, on the other hand, is insoluble in aqueous solutions in the absence of a carrier protein and its half-life in blood is less than 1 hour. Therefore, FTY720 may be a more potent therapeutic agent than S1P. Another S1P analog, VPC01091, also interacts with S1P receptors, but has the unusual property of being an agonist for S1P1 receptor and an antagonist for S1P3 receptor.
Sphingosine-1-phosphate (S1P) has been demonstrated to induce many cellular effects, including those that result in platelet aggregation, cell proliferation, cell morphology, tumor-cell invasion, endothelial cell chemotaxis and endothelial cell in vitro angiogenesis. For these reasons, S1P receptors are good targets for therapeutic applications such as wound healing and tumor growth inhibition.
Sphingosine-1-phosphate signals cells in part via a set of G protein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5 (formerly Edg-1, Edg-5, Edg-3, Edg-6, and Edg-8, respectively). These receptors share 50-55% identical amino acids and cluster with three other receptors (LPA1, LPA2, and LPA3 (formerly Edg-2, Edg-4 and Edg-7)) for the structurally related lysophosphatidic acid (LPA).
A conformational shift is induced in the G-Protein Coupled Receptor (GPCR) when the ligand binds to that receptor, causing GDP to be replaced by GTP on the α-subunit of the associated G-proteins and subsequent release of the G-proteins into the cytoplasm. The α-subunit then dissociates from the βγ-subunit and each subunit can then associate with effector proteins, which activate second messengers leading to a cellular response. Eventually the GTP on the G-proteins is hydrolyzed to GDP and the subunits of the G-proteins reassociate with each other and then with the receptor. Amplification plays a major role in the general GPCR pathway. The binding of one ligand to one receptor leads to the activation of many G-proteins, each capable of associating with many effector proteins leading to an amplified cellular response.
S1P receptors make good drug targets because individual receptors are both tissue and response specific. Tissue specificity of the S1P receptors is desirable because development of an agonist or antagonist selective for one receptor localizes the cellular response to tissues containing that receptor, limiting unwanted side effects. Response specificity of the S1P receptors is also of importance because it allows for the development of agonists or antagonists that initiate or suppress certain cellular responses without affecting other responses. For example, the response specificity of the S1P receptors could allow for an S1P mimetic that initiates platelet aggregation without affecting cell morphology.
Sphingosine-1-phosphate is formed as a metabolite of sphingosine in its reaction with sphingosine kinase and is stored in abundance in the aggregates of platelets where high levels of sphingosine kinase exist and sphingosine lyase is lacking. S1P is released during platelet aggregation, accumulates in serum, and is also found in malignant ascites. Biodegradation of S1P most likely proceeds via hydrolysis by ectophosphohydrolases, specifically the sphingosine 1-phosphate phosphohydrolases.
Angiogenesis
Orthopaedic regenerative medicine has focused on remodeling the microvascular network to prevent ischemia and aid in nutrient and oxygen delivery to sites of injury. An important process which has held great attention in the biomedical arena is angiogenesis. Angiogenesis refers to the growth of new blood vessels, specifically the sprouting of new capillaries from pre-existing vessels which produce new capillary networks. More than four billion dollars have been invested in research and development for angiogenesis based-medicines, establishing this field of study as one of the most heavily funded in history. Additionally, approximately 314 million patients in Western nations can benefit from angiogenesis-stimulating therapies. Hence, it is essential to understand this process and components involved.
In the initial stage of angiogenesis, diseased or injured tissues produce and release growth factors which diffuse into tissues within close proximity. Some of these factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor, granulocyte colony-stimulating factor, hepatocyte growth factor, transforming growth factor alpha, and several others. These proteins then bind to and activate specific receptors on endothelial cells. Upon activation, signal pathways are initiated in the endothelial cells which facilitate the production of enzymes. These enzymes create dissolved holes in the basement membrane of existing blood vessels. Endothelial cells then begin to proliferate and subsequently migrate via the dissolved holes of the blood vessels. Next, adhesion molecules, or integrins (αvβ3, αvβ5), facilitate the pulling of new blood vessel sprouts forward. Additional enzymes, called matrix metalloproteinases (MMPs), are created to dissolve the tissue in front of the sprouting vessel tip. These MMPs ensure that as the vessel extends, the tissue is remodeled around the vessel. Blood vessel tubes then begin to form due to sprouting endothelial cells. Once formed, these individual tubes connect to existing blood vessels to create blood vessel loops which can circulate blood. To ensure these newly formed blood vessel tubes are stabilized and functional, smooth muscle cells and pericytes are recruited and provide structural support, essentially allowing blood flow to occur.
Three different processes may contribute to the growth of new blood vessels: vasculogenesis, arteriogenesis, and angiogenesis. Vasculogenesis is the primary process responsible for growth of new vasculature during embryonic development and may play a yet-undefined role in mature adult tissues. It is characterized by differentiation of pluripotent endothelial cell precursors (hemangioblasts or similar cells) into endothelial cells that go on to form primitive blood vessels. Subsequent recruitment of other vascular cell types completes the process of vessel formation. The occurrence of vasculogenesis in mature organisms remains an unsettled issue. It is thought to be unlikely that this process contributes substantially to the new vessel development that occurs spontaneously in response to ischemia or inflammation as a response to growth factor stimulation.
Arteriogenesis refers to the appearance of new arteries possessing a fully developed tunica media. The process may involve maturation of pre-existing collaterals or may reflect de novo formation of mature vessels. Examples of arteriogenesis include formation of angiographically visible collaterals in patients with advanced obstructive coronary or peripheral vascular disease. All vascular cell types, including smooth muscle cells and pericytes, are involved. Arteriogenesis is the preferred type of neovascularization for purposes of restoring myocardial perfusion. Native arterial collateralization is a complex process that involves multiple levels of stimulators, inhibitors, and modulators. Therefore, the discovery of a drug molecule that induces therapeutic arteriogenesis, including the self-propagating cascade of proliferation, migration, and chemotaxis would be useful.
Angiogenesis is the process responsible for formation of new vessels lacking developed media. Examples of angiogenesis include capillary proliferation in wound healing or along the border of myocardial infarction. Angiogenesis can be stimulated by a number of growth factors including fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). Further, insulin-like growth factor-I (IGF-I) can stimulate proliferation of these cells and can induce VEGF secretion. These growth factors appear to exert their effort directly on endothelial cells and reports indicate that these effects may be mediated through specific integrin molecules (αvβ3 or αvβ5).
The occurrence of both angiogenesis and arteriogenesis has been demonstrated conclusively in a variety of animal models, as well as in patients with coronary disease. Thus, insufficient angiogenesis may lead to tissue ischemia and failure. The recent discovery of novel angiogenic molecules has initiated efforts to improve tissue perfusion via therapeutic angiogenesis. However, rational design of novel treatment strategies and potential drugs mandates a better understanding of the molecular mechanisms of angiogenesis.
Hematopoietic Stem Cells
Hematopoietic stem cells are multipotent stem cells that give rise to all the blood cell types including human CD34+ stem cell. The CD34 molecule is a cluster of differentiation molecules present on certain cells within the human body. It is a cell surface glycoprotein and functions as a cell-cell adhesion factor. It may also mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells. CD34 is also the name for the human gene that encodes the protein.
Cells expressing CD34 (CD34+ cell) are normally found in the umbilical cord and bone marrow as hematopoietic cells and tend to migrate from the blood stream to the bone marrow along a gradient of stromal derived factor-1 (SDF-1) where SDF-1 levels are high in the bone marrow and low in the peripheral blood. SDF-1 is a cytokine belonging to the chemokine family CXCL12. When a bone marrow transplant patient receives allogeneic UCB mononuclear cells via intravenous infusion, successful engraftment entails UCB stem cells taking up residence in the patient's bone marrow. A peripheral blood mononuclear cell is any blood cell having a round nucleus. Activation of the complement system in the transplant patient as part of the stress response elicited by chemoradiotherapy conditioning activates proteases in the marrow that reduce SDF-1 concentration. Low SDF-1 levels in the bone marrow tend to lessen homing and engraftment of allogeneic UCB CD34 stem cells. Because the numbers of CD34+ hematopoietic stem cells (HSC) in UCB is low, methods to enhance engraftment of this population of cells are needed. Non-embryonic UCB-derived stem cells are non-controversial (with approval by the Vatican and all religious groups), and offer the potential for “off the shelf” cell therapeutic products that are easier to obtain and faster to distribute than cumbersome individual adult directly-donated bone marrow and blood cells.
AMD3100 is a small-molecule CXCR4 chemokine antagonist known to enhance mobilization of stem cells for autologous transplantation in patients with non-Hodgkin's lymphoma (NHL) and multiple mycloma (MM). It is also used in some cases in conjunction with G-CSF administration, but must be administered at least several days later. AMD3100 is an inhibitor of the interaction between stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4.
Limitations of current management of vascular disease include re-occlusion and diffuse small vessel disease. Prior evidence links the level of circulating marrow-derived HSC, characterized by expression of CD133 and CD34, with the occurrence of ischemic vascular events. Human HSC which express CD34 and CD133 surface markers have been shown in models of acute and chronic ischemia to augment blood flow and prevent myocardial necrosis There is emerging evidence of age-related diminution in the number and function of marrow-derived CD34/133+ HSC in response to ischemia.
Cellular and molecular mechanisms underlying homing to the marrow microenvironment, a key requirement for successful allogeneic transplantation, is incompletely characterized. Data acquired to date indicates that administration of required patient pre-conditioning with chemoradiotherapy prior to allogeneic donor HSC infusion causes a stress response, including S1P release from circulating red blood cells (RBC) in the peripheral blood; and simultaneous release of proteases in the marrow that diminish SDF-1 concentrations. These two biologic sequelae of the stress response in vivo normally maximize egress of HSC out of the marrow niche.
S1P has been shown by this group and others to act on human CD34+ HSC or murine Lineage 1−/Sca1+/c-kit+ (LSK) as a chemotactic factor in the peripheral blood, mediating egress of HSC from the marrow. Furthermore, activation of S1P receptors augments CXCR4-mediated signal transduction induced by SDF-1. These effects are most likely mediated by both the S1P1 and S1P3 receptors expressed on both primitive and committed CD34+ HSC. SDF-1 regulates the trafficking of HSC. SDF-1 is the ligand for CXCR4, which had been considered for many years as its only receptor. Thus, the SDF-1-CXCR4 axis has a unique and important biological role.
Polymers
Poly (D, L-lactic-co-glycolic acid) (PLAGA) and poly(3-hydroxybutrate-co-3-hydroxyvalerate) (PHBV) are biodegradable and biocompatible polymers commonly used for tissue-engineered scaffolds. One can tailor the degradation rate of these polymers by altering the ratio of each component in the polymer composition, thereby rendering them suitable drug-release devices for both local and systemic delivery.
PLAGA is an FDA-approved copolymer of polylactide (PLA) and polyglycolide (PGA). PLA is a hydrophobic material with a degradation time greater than 24 months, which allows for great drug delivery potential. Through metabolic pathways, PLA degrades to lactic acid. PGA is a hydrophilic material and degrades at a faster rate, typically between 6 and 12 months, resulting in the glycolic acid byproduct. The polyester PLAGA degrades through hydrolysis and exhibits bulk degradation, releasing the non-toxic byproducts lactic acid and glycolic acid. Because of these acidic byproducts, local pH changes must be considered during PLAGA degradation. When used as a drug-delivery vehicle, variables such as molecular weight (Mw), copolymer composition, and crystallinity influence polymer degradation and the corresponding drug release kinetics.
PHBV is a polyester copolymer of hydroxybutyrate and hydroxyvalerate with adjustable processing and mechanical properties. By altering the copolymer composition and Mw, one can modify properties of PHBV. The accumulation of degradation products β-hydroxybutyric acid and hydroxyvaleric acid can thus be controlled.
There is a long felt need in the art for compositions and methods to enhance wound healing, organ and tissue repair, and mobilization and recruitment of stem and progenitor cells. The present invention satisfies these needs.